Team:Kyoto/Results
Results
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1. Virus and Viroid detection
2. Feeding RNAi for western flower thrips
3. Other application for cut flower maintenance
4. Biomolecular Production Platform
Production of homemade enzymes
In order to reduce the cost of one assay of LAMP/RT-LAMP[1.1], we produced homemade DNA polymerase,
and Reverse
Transcriptase. For production, we transformed the plasmids into E.coli, cultured them, and purified
them using Ni-NTA
beads. In a dialysis step we changed the buffers to optimize their activities, and especially for RT,
separately prepared
p66 and p51 were dimerized.
Transformation
The genes of interest were cloned into pET-11a and were transformed into BL21 (DE3). A lot of colonies
were observed in all the plates. The plasmid maps for each are as follows.
pMK715: DNA polymerase
pMK713: p66(RT)
pMK714: p51(RT)
pMK715: DNA polymerase
pMK713: p66(RT)
pMK714: p51(RT)
Protein expression & purification
Each LB culture was grown at 37 °C to the log-phase. When OD600 reaches 0.35-0.7, we added IPTG at the
final concentration of 0.5 mM. After 3 hours of induction, we cells were harvested and frozen in
liquid-N2. After the cell disruption by sonication, each protein was purified using Ni-NTA beads.
Samples were eluted by a four-step gradient of Imidazole. We observed the correct bands in the
SDS-PAGE gel (shown below). The arrowheads indicate the targeted proteins.
Dialysis
We changed the elution Buffer to the storage Buffer for the reaction. We put the purified proteins in
dialysis bags and incubated them o/n at 4 °C in 500 mL of storage buffer. For RT, we added purified p66
and p51 to the same tube and incubated them at -30 °C for 4 days to make a dimer.
Checking the activity
We performed the LAMP reaction and reverse transcription reaction to confirm that the purified
homemade enzymes had activities[1.2][1.3].
[Checking the activity of homemade DNA polymerase]
We performed the LAMP reaction using sequence E and its primer set, which already proved to be successful. We used BST3.0 purchased from NEB as positive control, water as negative controll, and various gradients of purified homemade BST DNA polymerase as in the following table.
We performed the LAMP reaction at 63 °C for 45 minutes using a qPCR machine and measured the
fluorescence of Evagreen each cycle. We performed electrophoresis of the samples after the reaction
and observed ladder-like bands ,which is characteristic of the LAMP reaction,in some lanes. From this
result, we confirmed that the homemade DNA polymerase has activity.
[Checking the activity of homemade RT]
In addition to our homemade DNA polymerase, we examined our homemade reverse transcriptase activity in vitro. We used total yeast DNA/RNA for this assay. We selected the intron-containing region of Rpl19A as a template for reverse transcription. If there is no reverse transcription, the genomic DNA including an intron is amplified by PCR, resulting in a band of 876 bp. On the other hand, if cDNA is correctly synthesized by reverse transcription, a band of 370 bp without an intron should appear. We carried out reverse transcription experiments under the following conditions, and then PCR.
As shown below, the cDNA band was successfully observed in an RT-dependent manner. From this result,
we concluded the activity of homemade RT.
(From left to right: 1-4: newly prepared homemade RT with different enzyme concentrations, 5: no
enzyme, 6: Bst3.0, 7: old homemade RT, 8: no template control)
[Checking the activity of homemade DNA polymerase]
We performed the LAMP reaction using sequence E and its primer set, which already proved to be successful. We used BST3.0 purchased from NEB as positive control, water as negative controll, and various gradients of purified homemade BST DNA polymerase as in the following table.
In addition to our homemade DNA polymerase, we examined our homemade reverse transcriptase activity in vitro. We used total yeast DNA/RNA for this assay. We selected the intron-containing region of Rpl19A as a template for reverse transcription. If there is no reverse transcription, the genomic DNA including an intron is amplified by PCR, resulting in a band of 876 bp. On the other hand, if cDNA is correctly synthesized by reverse transcription, a band of 370 bp without an intron should appear. We carried out reverse transcription experiments under the following conditions, and then PCR.
Cas12a-mediated LAMP/RT-LAMP assay against 2 viruses and a viroid
LAMP against DMV using BST 3.0 and homemade enzyme
We performed a LAMP reaction using a part of the DMV genome synthesized by IDT, using both BST3.0 and
a homemade DNA
polymerase[1.4]. From the result of electrophoresis, we confirmed that the LAMP reaction successfully
happened. This
indicates that the primer set is suitable for LAMP.
In addition, in the experiment above, isothermal buffer 2 that optimizes the reverse transcription
activity of BST 3.0
was used, but by changing this buffer 2 to buffer 1 that is optimized for DNA amplification, the
activity of DNA
polymerase was increased.
Cas12a-mediated LAMP assay against DMV
[Checking that this assay works with BST3.0 and homemade DNA polymerase]
In order to check that this Cas12a-mediated LAMP assay system works properly, LAMP reaction was
performed with the
following components. These samples were incubated at 63℃ for 50min.
After LAMP, we performed Cas12a fluorescent assay in the following procedure and this procedure was
not changed during
all the following experiments.
[Examination of the detection limit of the Cas12a-mediated LAMP assay against DMV]
In order to verify the sensitivity of the Cas12a-mediated LAMP assay, we performed Cas12a-mediated LAMP assay with different copy numbers of DMV target DNA, from 10^10 copies to 1 copy, under the following conditions. LAMP was conducted at 63 °C for 30 min.
After the assay, we checked the fluorescence of FAM with the naked eye under a blue light reader.
Since there was no
significant difference between these 12 wells, we also measured the value of FAM quantitatively by a
qPCR machine. From
the following graphs, we concluded that BST3.0 can detect up to one copy.
[Verify that assay works with BST2.0 warm start]
The purchased enzyme BST3.0 which we had been using was out of stock, so we switched to using BST2.0 warmstart as an alternative enzyme. We confirmed the function of this enzyme by the same protocol as before. However, the amount of the enzyme was changed from 1 unit to 8 units as recommended by NEB[1.5].
After the assay, we checked the fluorescence of FAM with the naked eye under a blue light reader.
Then, we also measured
the value of FAM quantitatively by a qPCR machine. However, as shown in the following graph, we only
observed the
detection of 10^10copies. Therefore, we changed the amount of dNTP and MgSO4 of mix, but we could not
raise the
detection limit at all.
LAMP reaction at 63℃ for [X]min ( X depends on each experiment. This time, X=50 )
↓
250 nM LbCas12a was first pre-incubated with 250 nM of specific (or non-specific) LbCas12a crRNAs in 1x Cas12 reaction Buffer for 30 min at 37 ◦C to assemble Cas12-crRNA ribonucleoprotein (RNP) complexes.
↓
The reaction was diluted 4 times with a NEB 1x binding buffer.
↓
For fluorescence-based detection, for Cas12a 250 nM and for Cas12b, 500 nM of fluorophore-quencher (FQ) reporter (5′-/56-FAM/TTATT/ 3IABKFQ/-3′, IDT) and 2 µl of the RT-LAMP reaction was added to the pre-assembled Cas12- sgRNA RNP complexes and incubated at 37 ◦C for 10–30 minbr
After the assay, we observed the fluorescence of FAM with the naked eye under a blue light reader.
Then, we measured
FAM values quantitatively using a qPCR machine. With both BST3.0 and the homemade DNA polymerase, we
were able to detect
about 10-15 fold fluorescence than the negative control. This confirms that the Cas12a-mediated LAMP
assay works both
with BST3.0 and with the homemade BST DNA polymerse.↓
250 nM LbCas12a was first pre-incubated with 250 nM of specific (or non-specific) LbCas12a crRNAs in 1x Cas12 reaction Buffer for 30 min at 37 ◦C to assemble Cas12-crRNA ribonucleoprotein (RNP) complexes.
↓
The reaction was diluted 4 times with a NEB 1x binding buffer.
↓
For fluorescence-based detection, for Cas12a 250 nM and for Cas12b, 500 nM of fluorophore-quencher (FQ) reporter (5′-/56-FAM/TTATT/ 3IABKFQ/-3′, IDT) and 2 µl of the RT-LAMP reaction was added to the pre-assembled Cas12- sgRNA RNP complexes and incubated at 37 ◦C for 10–30 minbr
In order to verify the sensitivity of the Cas12a-mediated LAMP assay, we performed Cas12a-mediated LAMP assay with different copy numbers of DMV target DNA, from 10^10 copies to 1 copy, under the following conditions. LAMP was conducted at 63 °C for 30 min.
The purchased enzyme BST3.0 which we had been using was out of stock, so we switched to using BST2.0 warmstart as an alternative enzyme. We confirmed the function of this enzyme by the same protocol as before. However, the amount of the enzyme was changed from 1 unit to 8 units as recommended by NEB[1.5].
Cas12a-mediated RT-LAMP assay against CSVd using actual RNA samples
from infected
plants
Since the Cas12a-mediated LAMP assay that detects the synthetic DNA sequence was working well, we
proceeded to the
Cas12a-mediated RT-LAMP assay to also detect RNA viruses.
In these experiments((3) and (4)), we aimed to detect CSVd and TSWV using RNA samples obtained from
actual infected
plants. We got these RNA samples from a collaborator. He kindly gave us two RNA samples of each.
Since BST polymerase 2.0 has reverse transcription activity, we used twice the amount of the enzyme to
perform the RT-
LAMP reaction. Since we were told to dilute the samples 20 times, we prepared a 3-step gradient of
samples: original
concentration, diluted 20 times, and a no-template one. For RT-LAMP reaction, samples in the following
table were
incubated at 63 °C for 30 minutes.
After the assay, we checked the fluorescence of FAM with the naked eye under a blue light reader. We
also measured the
value of FAM quantitatively by a qPCR machine, but as shown in the graph, sufficient fluorescence
could not be confirmed.
Cas12a-mediated RT-LAMP assay against TSWV using actual RNA samples
from infected plants
We also performed Cas12a-mediated RT-LAMP assay on TSWV samples[1.6]. For TSWV we prepared two types
of
crRNA, and we performed the RT-LAMP reaction by incubating the samples in the following table at 63 °C
for 30 min.
After the assay, we checked the fluorescence of FAM with the naked eye under a blue light reader. We
also measured the value of FAM quantitatively by a qPCR machine, but as shown in the graph, sufficient
fluorescence could not be confirmed this time ,either. The problems seem to be that the quality of the
RNA samples was not confirmed and the optimal conditions for BST2.0 warm start were not sufficiently
examined, so we would like to solve these in the future.
References
1.1 Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., and Hase, T.
(2000) "Loop-mediated
isothermal amplification of DNA", Nucleic Acids Res. 28, E63.
1.2 Ali, Z., Aman, R., Mahas, A., Rao, G.S., Tehseen, M., Marsic, T., Salunke, R., Subudhi, A.K., Hala, S.M., Hamdan, S.M., et al. (2020). iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2. Virus Res. 288, 198129.
1.3 Seckler, J.M., Howard, K.J., Barkley, M.D., and Wintrode, P.L. (2009) "Solution structural dynamics of HIV-1 reverse transcriptase heterodimer", Biochemistry 48, 7646–7655.
1.4 Lee, S.H., Baek, Y.H., Kim, Y.-H., Choi, Y.-K., Song, M.-S., and Ahn, J.-Y. (2016) "One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV", Front. Microbiol. 7, 2166.
1.5 New England Biolabs, "Loop-Mediated Isothermal Amplification", https://international.neb.com/applications/dna-amplification-pcr-and-qpcr/isothermal-amplification/loop-mediated-isothermal-amplification-lamp
1.6 Fukuta, S., Ohishi, K., Yoshida, K., Mizukami, Y., Ishida, A., and Kanbe, M. (2004) "Development of immunocapture reverse transcription loop-mediated isothermal amplification for the detection of tomato spotted wilt virus from chrysanthemum", J. Virol. Methods 121, 49–55.
1.2 Ali, Z., Aman, R., Mahas, A., Rao, G.S., Tehseen, M., Marsic, T., Salunke, R., Subudhi, A.K., Hala, S.M., Hamdan, S.M., et al. (2020). iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2. Virus Res. 288, 198129.
1.3 Seckler, J.M., Howard, K.J., Barkley, M.D., and Wintrode, P.L. (2009) "Solution structural dynamics of HIV-1 reverse transcriptase heterodimer", Biochemistry 48, 7646–7655.
1.4 Lee, S.H., Baek, Y.H., Kim, Y.-H., Choi, Y.-K., Song, M.-S., and Ahn, J.-Y. (2016) "One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV", Front. Microbiol. 7, 2166.
1.5 New England Biolabs, "Loop-Mediated Isothermal Amplification", https://international.neb.com/applications/dna-amplification-pcr-and-qpcr/isothermal-amplification/loop-mediated-isothermal-amplification-lamp
1.6 Fukuta, S., Ohishi, K., Yoshida, K., Mizukami, Y., Ishida, A., and Kanbe, M. (2004) "Development of immunocapture reverse transcription loop-mediated isothermal amplification for the detection of tomato spotted wilt virus from chrysanthemum", J. Virol. Methods 121, 49–55.
dsRNA production
Design
We conducted this experiment with the aim to silence the target gene by inducing RNAi by ds RNA
produced in E. coli. The target genes for this experiment are an essential gene of Frankliniella
occidentalis, a gene responsible for chloroplast formation during early development in Arabidopsis
thaliana, and a gene for a transcription factor that positively regulates PCD in Ipomoea nil.
Silencing these genes is expected to kill the Frankliniella occidentalis, whitening leaf of
Arabidopsis thaliana, and double the flowering time of Ipomoea nil [2.1][2.2][2.3].
In producing dsRNA in E. coli, the plasmid design was based on the following paper [2.4][2.5]. A plasmid containing the two target gene sequence assembled in different orientations in the region between the two T7 promoters transformed into E. coli strain HT115 (DE3), which has low RNase activity and expresses T7 polymerase. Transformed E. coli transcribe hairpin RNA.
In addition, the following paper was used as a reference for the purification of dsRNA from E. coli culture[2.6].
In producing dsRNA in E. coli, the plasmid design was based on the following paper [2.4][2.5]. A plasmid containing the two target gene sequence assembled in different orientations in the region between the two T7 promoters transformed into E. coli strain HT115 (DE3), which has low RNase activity and expresses T7 polymerase. Transformed E. coli transcribe hairpin RNA.
In addition, the following paper was used as a reference for the purification of dsRNA from E. coli culture[2.6].
Plasmid construction
Plasmid construction 1
Connect the L4440 which is treated by restriction enzyme with a part of the target gene by INFRONT
assembly.
As a result,the plasmid was not created correctly. It is thought that the plasmid left over when the vector was treated with restriction enzymes appeared as a colony. Therefore, we gave up on this method and tried a new method.
As a result,the plasmid was not created correctly. It is thought that the plasmid left over when the vector was treated with restriction enzymes appeared as a colony. Therefore, we gave up on this method and tried a new method.
Plasmid construction 2
Multiple restriction enzymes were combined to create the target plasmid.
I was able to confirm that the insertion of one fragment was successful. For 7~12, a band of about 1100
bp is a correct insertion of two inserts. c shows a fragment of the correct length, but other than
that, no bands of the correct length can be seen.
Therefore, 7~12 is examined by cutting it with another enzyme (Xho1).
If the correct insert had been inserted, two cuts would have occurred and two bands would have been visible. However, since only one band can be seen as a major band, it is thought that the second insert was not inserted correctly.
The three plasmids with one insert (pR015, pR016, and pR017) are considered complete, and the plasmid with two inserts (pR019) is considered provisionally complete.
(1insertion 1, 2 VATPase-B 3,4 Trxz-3 5,6 EPH1
2insertion 7,8,9 Trxz-3 10,11,12 VATPase-B )
Therefore, 7~12 is examined by cutting it with another enzyme (Xho1).
If the correct insert had been inserted, two cuts would have occurred and two bands would have been visible. However, since only one band can be seen as a major band, it is thought that the second insert was not inserted correctly.
(2insertion 1,2,3 Trx-Z 4,5,6 V-ATPase )
The three plasmids with one insert (pR015, pR016, and pR017) are considered complete, and the plasmid with two inserts (pR019) is considered provisionally complete.
RNA Production
HT115 (DE3) is made into competent cells by calcium chloride method and transformed with plasmids
(pR005, pR015, pR016, pR017, pR019).
Enough colonies could be confirmed.
Considering the priority of the experiment, HT115 transformed with pR015 was cultured in two 1L LB, one each with pR017 and pR005.
The culture was started at 37 °C and IPTG was added when OD exceeded 0.4 to induce RNA production for 4 hours[2.7]. E. coli was disrupted and precipitated with NaCl, and RNA contained in the supernatant was collected by ethanol precipitation. Based on the results of absorbance measurement of the recovered RNA, it was assumed that many impurities other than the target RNA were contained in the RNA solution.
Collected RNA by ethanol precipitation after phenol-chloroform treatment. Electrophoresis in acrylamide gel was performed to confirm whether the target RNA was produced.
There is a major band around 700 bp（▲）, which is considered to be the target product because the band visible in EPH1 is a little shorter than that of vATPase-B.
The yield was estimated based on the results of this electrophoresis.
Additional production of dsRNA for vATPase-B was also performed in 6L LB medium. The production method was the same protocol as the previous one. For recovery of dsRNA, since phenol chloroform treatment was found to be necessary in the previous production, E. coli was crushed with NaCl and then purified by phenol chloroform treatment.
We were able to see a band that could be considered as the target dsRNA in this preparation as well as the previous one. In addition, a band of similar length was also observed in the in vitro transcription system.
The production amount of dsRNA was examined based on the electrophoresis result.
In the case of production by E. coli, the yield was much higher the second time when the recovery method was simplified. There is a possibility that the amount lost by ethanol precipitation is much higher than expected. In addition, the accuracy of the results is questionable because some of the expected concentrations have changed significantly from the last time they were electrophoresed. Compared to the in vitro transcription system, the production in E. coli is considered to be more cost effective.
Enough colonies could be confirmed.
Considering the priority of the experiment, HT115 transformed with pR015 was cultured in two 1L LB, one each with pR017 and pR005.
The culture was started at 37 °C and IPTG was added when OD exceeded 0.4 to induce RNA production for 4 hours[2.7]. E. coli was disrupted and precipitated with NaCl, and RNA contained in the supernatant was collected by ethanol precipitation. Based on the results of absorbance measurement of the recovered RNA, it was assumed that many impurities other than the target RNA were contained in the RNA solution.
Collected RNA by ethanol precipitation after phenol-chloroform treatment. Electrophoresis in acrylamide gel was performed to confirm whether the target RNA was produced.
(1: VATPase1, 2: VATPase2, 3: EPH1, 4: L4440 5: VATPase1, 6: VATPase2, 7: EPH1, 8: L4440)
There is a major band around 700 bp（▲）, which is considered to be the target product because the band visible in EPH1 is a little shorter than that of vATPase-B.
The yield was estimated based on the results of this electrophoresis.
Additional production of dsRNA for vATPase-B was also performed in 6L LB medium. The production method was the same protocol as the previous one. For recovery of dsRNA, since phenol chloroform treatment was found to be necessary in the previous production, E. coli was crushed with NaCl and then purified by phenol chloroform treatment.
1.L4440 (add production) 1ug 2 vATPase-B add production (7ug) 3 vATPase-B(add
production)（1ug）4 vATPase-B-1 (1ug）5 vATPase-B-2 (1ug), in vitro RNA production（6 vATPase-B,7 Trxz,8
EPH1）1ug each)
We were able to see a band that could be considered as the target dsRNA in this preparation as well as the previous one. In addition, a band of similar length was also observed in the in vitro transcription system.
The production amount of dsRNA was examined based on the electrophoresis result.
In the case of production by E. coli, the yield was much higher the second time when the recovery method was simplified. There is a possibility that the amount lost by ethanol precipitation is much higher than expected. In addition, the accuracy of the results is questionable because some of the expected concentrations have changed significantly from the last time they were electrophoresed. Compared to the in vitro transcription system, the production in E. coli is considered to be more cost effective.
Thrips assay
Overview
Insect pests are one of the major problems in the production stage of the flower industry. In
particular, western flower thrips feed on a variety of flowers such as roses and carnations, and have
been selected as one of “The 100 most invasive alien species” in Japan. In addition, it transmits
Tomato spotted wilt virus (TSWV), a virus that has become a serious problem in production along with
pests.
We have demonstrated that feeding dsRNA targeting essential genes of western flower thrips can induce RNAi and kill the pest. The dsRNA used in this assay was produced by E. coli.
We have demonstrated that feeding dsRNA targeting essential genes of western flower thrips can induce RNAi and kill the pest. The dsRNA used in this assay was produced by E. coli.
Assay method and results
Specifically, 200 µl of a 200 ng/µl dsRNA solution targeting vATPase-B (Milli-Q was used as a negative
control) was pipetted in a PCR tube and the petiole of a green bean primary leaf was
immersed[3.1][3.2][3.3]. After 2
hours, three leaf fragments of 1 cm × 2 cm were cut from the soaked bean leaves and placed in three
petri dishes with about 10 thrips. The mortality and leaf damage were checked after 24, 48, 72, and 96
hours.
The results are shown below.
In particular, there was a large difference in the mean mortality rate between dsRNA-treated and negative control thrips on day 3: 8.3% and 31.3%, respectively. This result confirms the insecticidal effect of dsRNA indirectly feeding to the western flower thrips.
The results are shown below.
In particular, there was a large difference in the mean mortality rate between dsRNA-treated and negative control thrips on day 3: 8.3% and 31.3%, respectively. This result confirms the insecticidal effect of dsRNA indirectly feeding to the western flower thrips.
Determination of whitening rate in Arabidopsis
Overview
Endogenous genes in plants play a major role in flower senescence. If the genes involved in floral
senescence can be knocked down using external dsRNA, it is expected that floral senescence will be
suppressed and flowers will last longer.
We used a model plant, Arabidopsis thaliana, to see if RNAi could be induced by dsRNA solutions
targeting an important endogenous gene. The dsRNA used in this assay was synthesized by in vitro
transcription.
Assay method and results
Specifically, 200 µl of 0.1, 1, or 10 ng/l dsRNA solution targeting Trxz (Milli-Q was used as a
negative control) was placed in a PCR tube, and Arabidopsis roots were immersed. For this assay, Dr.
Shimada provided us with Arabidopsis plants that had sprouted after being sown on the plate on August
31.
The condition of Arabidopsis after one week is shown below.
As a result, the RNAi effect was not well observed as a whole. In this study, the RNAi assay was performed by calculating the concentration of dsRNA obtained by in vitro transcription from the values measured by NanoDrop. However, electrophoresis showed that only a small amount of dsRNA in the target band was included. Therefore, the low concentration of dsRNA is considered to be the reason why the RNAi effect was not seen well.
The condition of Arabidopsis after one week is shown below.
From the left lane, negative control, Arabidopsis soaked in 0.1, 1, and 10
ng/L dsRNA solution (three replicates)
From the left lane, negative control, Arabidopsis soaked in 0.1, 1, and 10
ng/L dsRNA solution (three replicates)
As a result, the RNAi effect was not well observed as a whole. In this study, the RNAi assay was performed by calculating the concentration of dsRNA obtained by in vitro transcription from the values measured by NanoDrop. However, electrophoresis showed that only a small amount of dsRNA in the target band was included. Therefore, the low concentration of dsRNA is considered to be the reason why the RNAi effect was not seen well.
References
2.1 Andongma, A.A., Greig, C., Dyson, P.J., Flynn, N., and Whitten, M.M.A. (2020) "Optimization of
dietary RNA interference
delivery to western flower thrips Frankliniella occidentalis and onion thrips Thrips tabaci",
Arch. Insect Biochem. Physiol.
103, e21645.
2.2 Chen, Z., He, J., Luo, P., Li, X., and Gao, Y. (2018). Production of functional double-stranded RNA using a prokaryotic expression system in Escherichia coli. Microbiologyopen e787.
2.3 Shibuya, K., Shimizu, K., Niki, T., and Ichimura, K. (2014) "Identification of a NAC transcription factor, EPHEMERAL1, that controls petal senescence in Japanese morning glory", Plant J. 79, 1044–1051.
2.4 Tenllado, F., Martínez-García, B., Vargas, M., and Díaz-Ruíz, J.R. (2003) "Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections", BMC Biotechnol. 3, 3.
2.5 Timmons, L., and Fire, A. (1998) "Specific interference by ingested dsRNA", Nature 395, 854.
2.6 https://2019.igem.org/Team:SZU-China/Experiment.
2.7 Papić, L., Rivas, J., Toledo, S., and Romero, J. (2018) "Double-stranded RNA production and the kinetics of recombinant Escherichia coli HT115 in fed-batch culture", Biotechnol Rep (Amst) 20, e00292.
3.1 Andongma, A.A., Greig, C., Dyson, P.J., Flynn, N., and Whitten, M.M.A. (2020) "Optimization of dietary RNA interference delivery to western flower thrips Frankliniella occidentalis and onion thrips Thrips tabaci. Arch. Insect Biochem. Physiol. 103, e21645.
3.2 Han, S.H., Kim, J.H., Kim, K., and Lee, S.H. (2019) "Selection of lethal genes for ingestion RNA interference against western flower thrips, Frankliniella occidentalis, via leaf disc-mediated dsRNA delivery. Pestic", Biochem. Physiol. 161, 47–53.
3.3 Singh, S., Gupta, M., Pandher, S., Kaur, G., Goel, N., Rathore, P., and Palli, S.R. (2019) "RNA sequencing, selection of reference genes and demonstration of feeding RNAi in Thrips tabaci (Lind.) (Thysanoptera: Thripidae)", BMC Mol. Biol. 20, 6.
2.2 Chen, Z., He, J., Luo, P., Li, X., and Gao, Y. (2018). Production of functional double-stranded RNA using a prokaryotic expression system in Escherichia coli. Microbiologyopen e787.
2.3 Shibuya, K., Shimizu, K., Niki, T., and Ichimura, K. (2014) "Identification of a NAC transcription factor, EPHEMERAL1, that controls petal senescence in Japanese morning glory", Plant J. 79, 1044–1051.
2.4 Tenllado, F., Martínez-García, B., Vargas, M., and Díaz-Ruíz, J.R. (2003) "Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections", BMC Biotechnol. 3, 3.
2.5 Timmons, L., and Fire, A. (1998) "Specific interference by ingested dsRNA", Nature 395, 854.
2.6 https://2019.igem.org/Team:SZU-China/Experiment.
2.7 Papić, L., Rivas, J., Toledo, S., and Romero, J. (2018) "Double-stranded RNA production and the kinetics of recombinant Escherichia coli HT115 in fed-batch culture", Biotechnol Rep (Amst) 20, e00292.
3.1 Andongma, A.A., Greig, C., Dyson, P.J., Flynn, N., and Whitten, M.M.A. (2020) "Optimization of dietary RNA interference delivery to western flower thrips Frankliniella occidentalis and onion thrips Thrips tabaci. Arch. Insect Biochem. Physiol. 103, e21645.
3.2 Han, S.H., Kim, J.H., Kim, K., and Lee, S.H. (2019) "Selection of lethal genes for ingestion RNA interference against western flower thrips, Frankliniella occidentalis, via leaf disc-mediated dsRNA delivery. Pestic", Biochem. Physiol. 161, 47–53.
3.3 Singh, S., Gupta, M., Pandher, S., Kaur, G., Goel, N., Rathore, P., and Palli, S.R. (2019) "RNA sequencing, selection of reference genes and demonstration of feeding RNAi in Thrips tabaci (Lind.) (Thysanoptera: Thripidae)", BMC Mol. Biol. 20, 6.
Peptide production
Design
The following experiments were conducted to reduce the number of bacteria that cause vessel blockage
and prolong the life of flowers by producing antibacterial peptides in E. coli and adding them to
vases.
We also produced and added a peptide called NOP1, which inhibits ethylene signaling, in the same way to prolong the life of cut flowers from a different perspective. The following paper was used as a reference to produce the peptide[4.1][4.2][4.3].
The peptide is expressed as a fusion protein: peptide-Mxe GyrA intein-linker-ELK16. The fusion protein aggregates and precipitates due to the action of ELK16. When DTT is added to the precipitated fusion protein, self-cleavage occurs at the N-terminus of the intein, and the peptide is cut out into the water layer. To produce the fusion protein, a plasmid was created by assembling the gblock encoding the fusion protein and pet-11a. The following three types of antimicrobial peptides were used: Defensin-1, Cecropin-A, LL37[4.4][4.5][4.6].
We also produced and added a peptide called NOP1, which inhibits ethylene signaling, in the same way to prolong the life of cut flowers from a different perspective. The following paper was used as a reference to produce the peptide[4.1][4.2][4.3].
The peptide is expressed as a fusion protein: peptide-Mxe GyrA intein-linker-ELK16. The fusion protein aggregates and precipitates due to the action of ELK16. When DTT is added to the precipitated fusion protein, self-cleavage occurs at the N-terminus of the intein, and the peptide is cut out into the water layer. To produce the fusion protein, a plasmid was created by assembling the gblock encoding the fusion protein and pet-11a. The following three types of antimicrobial peptides were used: Defensin-1, Cecropin-A, LL37[4.4][4.5][4.6].
Peptide production
Plasmid construction
As shown in the figure, PET11a was cut with restriction enzymes (BamH1, Nde1), and gblocks coding the
sequences of fusion protein connected by INFRONT assembly. The plasmid is completed as designed.
Peptide production
The production of peptides was carried out with reference to[4.3].
The plasmids from pR001 to pR004 were transformed into E. coli BL21 and colonies were formed on the
plate.
Each colony was transferred to 1L LB medium for culture and production of peptide.
The culture was started at 37 °C. The OD was measured sometimes, and when the OD exceeded 0.35, IPTG
was used to induce peptide expression. E. coli was collected as a precipitate after incubation.
Def1 looked a little reddish, unlike the other precipitations. This may be the color of the expressed
Def1.
E. coli collected as precipitate was suspended in BufferB1 (20 mM TrisHCl 8.5, 500 mM NaCl, 1 mM EDTA) and disrupted by French pressing twice and sonication (10 sonicate, 1 min) six times. The precipitate containing the fusion protein was collected by centrifugation. The precipitates were washed by suspending them in Buffer B1, centrifuging them, and discarding the supernatant five times to remove the soluble components in the precipitates. The precipitates were divided equally, and Buffer B3 (20 mM TrisHCl 8.5, 500 mM NaCl, 1 mM EDTA 40 mM DTT) was added to one of the precipitates, and peptides were cut out by cleavage of inteins for one whole day. The other half of the collected precipitates were sonicated through water (2s sonication, 2s rest) x 100 or normal sonication (2s sonication, 2s rest) x 100 for additional disruption. The precipitates were then washed by suspending them in BufferB1, centrifuging and discarding the supernatant, repeated three times. After washing, the precipitates were mixed with buffer B3 to separate the peptides from the precipitates.
E. coli collected as precipitate was suspended in BufferB1 (20 mM TrisHCl 8.5, 500 mM NaCl, 1 mM EDTA) and disrupted by French pressing twice and sonication (10 sonicate, 1 min) six times. The precipitate containing the fusion protein was collected by centrifugation. The precipitates were washed by suspending them in Buffer B1, centrifuging them, and discarding the supernatant five times to remove the soluble components in the precipitates. The precipitates were divided equally, and Buffer B3 (20 mM TrisHCl 8.5, 500 mM NaCl, 1 mM EDTA 40 mM DTT) was added to one of the precipitates, and peptides were cut out by cleavage of inteins for one whole day. The other half of the collected precipitates were sonicated through water (2s sonication, 2s rest) x 100 or normal sonication (2s sonication, 2s rest) x 100 for additional disruption. The precipitates were then washed by suspending them in BufferB1, centrifuging and discarding the supernatant, repeated three times. After washing, the precipitates were mixed with buffer B3 to separate the peptides from the precipitates.
Electrophoresis on Tricine SDSPAGE
In order to confirm the actual existence of the peptides, we performed electrophoresis with Tricine
SDSPAGE. Tricine SDSPAGE can detect proteins with smaller molecular weight than normal SDSPAGE. The
results of the electrophoresis were as follows.
The band that is considered to be correct in Def1 (5713 Da) appears, while it could not be confirmed as a major
band in the rest of the peptides. Also, the mass of the fusion protein of intein and ELK16 is about 25
kDa, and this band is also visible. It seems that the impurities have been reduced by additional
sonication, but still many impurities remained.
An ultrafiltration was performed to remove impurities above 10 kDa for HPLC and assays.
New plasmid construction
There are three problems that we found during the first round of peptide purification. First, when the
fusion protein is recovered as a precipitate using ELK16, the purified peptide contains many
impurities
even after washing.Second, the precipitates contain highly viscous material such as genomic DNA,
which
makes it difficult to repeat peptide production because of the long time required to wash the
precipitates. Third, peptides other than Def1 cannot be identified as major bands, and the amount
recovered is quite low. As a second peptide production strategy to solve these problems, we used a
system that uses His tag and intein. The peptide-containing fusion protein can be recovered in a
column
by using the binding of His tag to nickel beads.
Using an additional primer, a DNA fragment of His tag-intein-peptide was amplified by PCR from the
gblock of ELK16-intein-peptide. INFRONT assembly was done to create a plasmid.
N end rule
The effect of n-end-rule was also considered as the cause of the problem of low recovery[4.7].
About E. coli, the following rules exist
Proteins which have Arginine, lysine, leucine, phenylalanine, tyrosine, and tryptophan at the amino
terminal have a half-life of two minutes. On the other hand, other peptides have a half-life of more
than 10 hour.
In the plasmid we created, only Def1 has a long half-life, while CecA, LL37, and NOP1 have short half-lives. I thought that this might have been the reason for the low recovery of peptides other than Def1, so we designed to improve the N-terminal of the peptides. Create two plasmids, one for Histag purification and one for ELK16 purification, for the production of NOP1 peptides with a Valine at the N-terminal end.
In the plasmid we created, only Def1 has a long half-life, while CecA, LL37, and NOP1 have short half-lives. I thought that this might have been the reason for the low recovery of peptides other than Def1, so we designed to improve the N-terminal of the peptides. Create two plasmids, one for Histag purification and one for ELK16 purification, for the production of NOP1 peptides with a Valine at the N-terminal end.
Second peptide production
Plasmid construction
PCR was used to create a DNA fragment of fusion protein include NOP1v.INFRONT assembly was done to
create a plasmid.
In plasmid construction, pR009 to pR014 were completed.
Peptide production
The plasmid for His tag purification and the control pet11a were transformed into BL21. From the
colonies on the plate, we cultured them in 1L of LB medium at 37C. After IPTG induction, the culture
was
transferred to 30C for 6 hours and the E. coli that produced the peptide was collected as a
precipitate.
The cultured E. coli was crushed by sonication and washed with bufferA. The target protein was eluted with Ni-NTA beads at four concentrations of Imidazole: 70 mM, 95 mM, 120 mM, and 270 mM.
Purification of proteins using the His-tag was more successful than using ELK16
After purification of fusion protein, add DTT for these to cut out peptides.
The peptides were electrophoresed by Tricine SDS PAGE, and the bands of the peptides could be seen in
Def1 with and without DTT. In all lanes except pet11a, a band (of 24 kDa) that seems to be HIs-intein
is
visible. Therefore, the expression of the fusion protein and the purification of the fusion protein
using His tag seems to have been successful. There are three possible reasons why the other peptides
were not identified as bands. First, NOP1 is a particularly short peptide and may have been run away
during electrophoresis. The second possibility is that the peptides were not cleaved by the
self-cleavage of the intein, and the third possibility is that the intein was cleaved at some point
before purification and moved to another fraction during imidazole elution. Def1 was also cut out
before
The cultured E. coli was crushed by sonication and washed with bufferA. The target protein was eluted with Ni-NTA beads at four concentrations of Imidazole: 70 mM, 95 mM, 120 mM, and 270 mM.
peptide (concentration of Imidazole)
1.NOP1v(270 mM) 2.NOP1v(120mM) 3.NOP1(270mM) 4.NOP1(120mM) 5.Def1(270mM) 6.Def1(120mM)
7.CecA(270 mM)
8.CecA(120 mM) 9. LL37(270 mM) 10.LL37(120 mM)11.Pet11a(270 mM) 12.Pet11a(120 mM)
(1 CecA 2 Def1 3 LL37 4 NOP1 5 NOP1v 6 pet11a 7 CecA 8 Def1 9 LL37 10 NOP1 11 NOP1v)
MS
Mass spectrometry was performed by Bruker Daltonics flexAnalysis. 0.5 µl of sample was mixed with 0.5 µl
of Matrix solution and air-dried. After that, the plate was set and calibrated by standard and
insulin,
and the measurement was started. The results are shown below. The possible molecular weights are shown
in
the table below.
However, we did not observe the expected molecular weights for all peptides, which is questionable since the Tricine SDS PAGE data suggests that the fusion proteins themselves were produced. It is possible that the excised peptides have undergone unnecessary modification or cleavage in the cell, or that they have been partially degraded.
PDF ( click to expand PDF viewer )
However, we did not observe the expected molecular weights for all peptides, which is questionable since the Tricine SDS PAGE data suggests that the fusion proteins themselves were produced. It is possible that the excised peptides have undergone unnecessary modification or cleavage in the cell, or that they have been partially degraded.
Anti-Bacterial system assay
Overview
One of the major causes of wilting cut flowers is that bacteria growing in the vase invades the vessel
and prevents flowers from absorbing water. Solving this problem, we decided to use antimicrobial
peptides from organisms to inhibit growth of them. Although there are many kinds of antimicrobial
peptides, not all of them are capable of attacking all species of bacteria. Considering that, we tried
to analyze what kinds of bacteria existed in the vase and use some antimicrobial peptides to kill them
effectively.
To show that, we had grown flowers for one week. We took water from each sample and spread it on the standard agar medium（Agar 15g Glucose1g peptone 6g Yeast extarct2.5g per 1L）in order to observe the changes in bacterial flora. Besides, we did the disk diffusion in order to see if the antimicrobial peptides we purified could actually inhibit the growth of bacteria.
KeyAchievement : We found that the bacterial flora of the vase had changed as the days went by. The antimicrobial peptides we purified did not show the antimicrobial activity.
To show that, we had grown flowers for one week. We took water from each sample and spread it on the standard agar medium（Agar 15g Glucose1g peptone 6g Yeast extarct2.5g per 1L）in order to observe the changes in bacterial flora. Besides, we did the disk diffusion in order to see if the antimicrobial peptides we purified could actually inhibit the growth of bacteria.
KeyAchievement : We found that the bacterial flora of the vase had changed as the days went by. The antimicrobial peptides we purified did not show the antimicrobial activity.
The observation of the bacterial flora of the vase water
Material and method
We choose carnation and rose that are famous for and delphinium that the florist recommends as a
flower
that trends to cloud the water. We had grown them for one week without changing their water. We took
50 µl water from each of the stem, the vase surface, and water for that one week and spread it on the
standard agar medium by conlarge stick. When we smeared water samples, we diluted them as there were
about 1000 colonies according to each OD600. Also, this one week, we measured pH. The water was
absorbed
by flowers or evaporated, so we added water and kept 30mL for one week.
We did the same thing twice as a preliminary experiment. From the results of them, the species of
bacteria became similar after about one week. So, in this experiment, I decided to observe them for a
week.
Changes in water in the vase over time
Fig.1 shows the results of the normal tap water changes in bacterial flora. As it shows, the tap water
from day1 to day4 could not grow any bacteria, but from day5 to day7, many kinds of bacteria rapidly
increased.
Fig.1-Changes in pH and bacterial flora of normal tap water
Fig.2-OD600 and dilution rate of normal tap water
Changes in bacterial flora focusing on each flower or the place
From Fig.3, as for rose, the red bacteria appeared on the day4 in the vase water. After that, the
green one became dominant. Taking into account the pH change, it increased on day4 with an increase of
red one and decreased on day5 with an increase of green one.
Although the bacterial amount in the vase of carnation was so large compared to other flowers, the clear bacterial change would not show. The similar bacteria appeared in the stem and the side of vase. Besides, the bacteria in carnation would grow earlier than two.
As for delphinium, the red bacteria appeared at the same time pH increased. After that, when the yellow one was found, pH decreased.
In this experiment, in both rose and delphinium, the red bacteria that we had not seen before appeared
on the day4, but on the day5, they disappeared. We did not know if these two red bacteria were the
same. However, we think that what is important would be that the bacterial flora in the vase between
on the early day and the later was different.
We think that the three factors would cause this trend. First, the many kinds of bacteria in the air could fall into the vase that we set up. In this experiment, we did not put the vase in the sterile condition to recreate the situation of growing flowers at home. So, the bacteria or dust from the air could accumulate in the vase and change its bacterial flora.
Second, although it is thought that this cause might be more influential, the secretion from the flower could make the vase water more nutrient, which changed the environment in the vase. Although there were no bacteria in the early vase water, on the day6 or 7, the water had been so dirty you could see it. The degree or rate of contamination varied by species, which could show that the composition of vase water could rely on the kind or amount of secretion from flowers. This would change the water continuously and lead to the variation of bacteria that adapt and grow in each water environment.
Third, a more interesting possibility is that environmental changes that the previous bacteria had caused prevented the growth of the bacteria that was previously the dominant one[5.3]. Such changes in dominant species, which have been well studied in the case of plankton in natural lakes, are observed in a variety of cases. It is thought that this is partly due to environmental changes as a result of the flourishing of dominant species, such as the depletion and decomposition of their preferred nutrients and the accumulation of waste products that hinder their growth. In this experiment, we also noted that the pH of the water increased at the same time as the red colony bacteria increased. There is no evidence that the red colony is directly responsible for this pH change. However, we observed that at least one environmental factor changed during the change of the dominant species.
From these experiments we have learned that the flora of the water in the vase is dynamically changing. This led us hypothesize that in order to suppress the environmental changes in the vase water in advance and to make flowers live longer consequently, it would be more effective to use antimicrobial substances to the bacterium in relatively clean water on the second or third day of the experiment than to use them to the bacterium in dirty water on the seventh day.
In this experiment, we also examined the flora of samples collected from different parts of the vase. The results showed that the bacterium did not grow on stems or the vase surface first, but rather moved from the water to the stems and vase surface. This suggests that antibacterial substances should be added to the water first, before they are added to the stems and vases. It might be an effective way to investigate the initial bacterium appearing in the water and to use the antibacterial substances to them.
Fig.3-Changes in bacterial flora and pH of rose
Fig.4-OD600 and dilution rate of rose
Fig.5-Changes in bacterial flora and pH of carnation
Fig.6-OD600 and dilution rate of carnation
Fig.7-Changes in bacterial flora and pH of delphinium
Fig.8-OD600 and dilution rate of carnation
We think that the three factors would cause this trend. First, the many kinds of bacteria in the air could fall into the vase that we set up. In this experiment, we did not put the vase in the sterile condition to recreate the situation of growing flowers at home. So, the bacteria or dust from the air could accumulate in the vase and change its bacterial flora.
Second, although it is thought that this cause might be more influential, the secretion from the flower could make the vase water more nutrient, which changed the environment in the vase. Although there were no bacteria in the early vase water, on the day6 or 7, the water had been so dirty you could see it. The degree or rate of contamination varied by species, which could show that the composition of vase water could rely on the kind or amount of secretion from flowers. This would change the water continuously and lead to the variation of bacteria that adapt and grow in each water environment.
Third, a more interesting possibility is that environmental changes that the previous bacteria had caused prevented the growth of the bacteria that was previously the dominant one[5.3]. Such changes in dominant species, which have been well studied in the case of plankton in natural lakes, are observed in a variety of cases. It is thought that this is partly due to environmental changes as a result of the flourishing of dominant species, such as the depletion and decomposition of their preferred nutrients and the accumulation of waste products that hinder their growth. In this experiment, we also noted that the pH of the water increased at the same time as the red colony bacteria increased. There is no evidence that the red colony is directly responsible for this pH change. However, we observed that at least one environmental factor changed during the change of the dominant species.
From these experiments we have learned that the flora of the water in the vase is dynamically changing. This led us hypothesize that in order to suppress the environmental changes in the vase water in advance and to make flowers live longer consequently, it would be more effective to use antimicrobial substances to the bacterium in relatively clean water on the second or third day of the experiment than to use them to the bacterium in dirty water on the seventh day.
In this experiment, we also examined the flora of samples collected from different parts of the vase. The results showed that the bacterium did not grow on stems or the vase surface first, but rather moved from the water to the stems and vase surface. This suggests that antibacterial substances should be added to the water first, before they are added to the stems and vases. It might be an effective way to investigate the initial bacterium appearing in the water and to use the antibacterial substances to them.
Disk Diffusion method
The result of the disk diffusion method
NOP-1 assay
Assay 1
Materials and Methods
To research the effect of NOP-1 and NOP-1v synthesized by E.coli, we conducted this assay. We used
potted Dianthus that has an ethylene-dependent pathway.
We didn’t use cut flowers to avoid prior treatment with STS. Following previous research, We used 1mM
AgNO3 aq as Positive control and the same buffer for NOP-1 as Negative control[6.1]. Also, We
confirmed the
effect of BL and Brz that Professor Nakano gave us[6.2]. We used the same buffer for BL or Brz as
Negative
control.
The Result of Assay 1
Note that the start date of this assay should be the 1st day.
The effect on life prolongation byNOP-1 and NOP-1v synthesized by E.coli
As shown above, NOP-1 and NOP-1v extend the flower life by 1-2 days against the Negative control. And
it seemed to have the same life prolongation as 1mM AgNO3 used as the positive control.
The effect on life prolongation by BL and Brz Professor that Nakano gave us
As shown above, Brz extended the life of flowers for two days against the negative control. And it
seemed to have the same life prolongation as 1mM AgNO3 used as the positive control. In addition, BL
did not have the same life prolongation as Brz. These results were similar to the findings of
Professor Nakano, who gave us BL and Brz.
NOP-1, NOP-1v, and Brz seemed to have life prolongation of Dianthus. However, the number of each sample was only one. So we were not able to conclude the effects, and we retried to conduct an assay.
The effect on life prolongation byNOP-1 and NOP-1v synthesized by E.coli
The effect on life prolongation by BL and Brz Professor that Nakano gave us
NOP-1, NOP-1v, and Brz seemed to have life prolongation of Dianthus. However, the number of each sample was only one. So we were not able to conclude the effects, and we retried to conduct an assay.
Assay 2
Materials and Methods
To confirm the result of assay 1, we increased the number of samples and retried an assay. We also
researched the effect of chemical synthesized NOP-1. We used the same buffer for NOP-1 as the Negative
control.
The Result of Assay 2
Note that the start date of this assay should be the 1st day.
the effect on long prolongation by NOP-1 and NOP-1v synthesized by E.coli
the effect on long prolongation by chemical synthesized NOP-1
Both NOP-1 and NOP-1v in vivo and NOP-1 in vitro didn't show life prolongation.
the effect on long prolongation by NOP-1 and NOP-1v synthesized by E.coli
Biofilm degrading enzyme experiment
Overview
We had thought of antimicrobial peptides as the main countermeasure against bacteria in vases.
However, after consulting with Dr. Nomura, we learned that even in a vase, bacteria are likely to form
biofilms to protect themselves from antimicrobials.
Therefore, we decided to investigate the effect of biofilm-degrading enzymes on flowers by collaborating with the iGEM team of Gunma University, with whom we had formed a partnership.
Key achievements : The ability of the enzyme could not be confirmed.
Therefore, we decided to investigate the effect of biofilm-degrading enzymes on flowers by collaborating with the iGEM team of Gunma University, with whom we had formed a partnership.
Key achievements : The ability of the enzyme could not be confirmed.
Assay
Preparing dahlias and asters
We went to Hanachu florist near Kyoto University, and bought flowers based on the diameter of their
stems: thick and thin. We chose dahlias and asters as a sample. Each of them was put in a vase filled
with 200 mL of tap water and left them at room temperature for three days.
Check the activity of BF enzyme
After 3 days, flowers were removed from the vase and cut into 1cm, 2cm, and 3cm pieces from the tip.
Each flower was cut into two along the diameter of the face and one was used as the control group and
the other as the experimental group.
Put the stems into a 1.5ml tube, add PBS, and shake it. Centrifuge and remove the PBS with an
aspirator. This was done a total of three times (3000 rpm). Then the control group was treated with
200 µl of PBS, and the study group was treated with 200 µl of Buffer + 10 µl of enzyme. After adding
the reagent, it was incubated overnight at 37℃.
Enzyme samples from Gunma Univ. require a dedicated buffer. Prepared the reagent listed below and PBS was added to become 10mL.
Enzyme samples from Gunma Univ. require a dedicated buffer. Prepared the reagent listed below and PBS was added to become 10mL.
qPCR
Nucleic acids were extracted from the bacteria by processing the samples with lysozyme and Protein K
incubated overnight. Nucleic acids were purified by phenol and chloroform, and the DNA retrieved by
ethanol precipitation was subjected to qPCR. The genomic DNA of DH5alpha, which was measured as a
standard, was amplified and a quantitative line was generated. The results are shown below. However,
we could not obtain any data for the sample because it was below the detection limit.
This may be due to the fact that the activity of the enzyme was weaker than expected and the fungus did not fall off from the stem, or it fell off when washed in PBS. It is also possible that the BF produced on the flowers could not be degraded by the α-galactosidase of Gunma University.
This may be due to the fact that the activity of the enzyme was weaker than expected and the fungus did not fall off from the stem, or it fell off when washed in PBS. It is also possible that the BF produced on the flowers could not be degraded by the α-galactosidase of Gunma University.
qPCR for genomic DNA of DH5alpha
References
4.1 Hoppen, C., Müller, L., Albrecht, A.C., and Groth, G. (2019) "The NOP-1 peptide derived from
the central regulator of
ethylene signaling EIN2 delays floral senescence in cut flowers", Sci. Rep. 9, 1287.
4.2 Wang, M., Zheng, K., Lin, J., Huang, M., Ma, Y., Li, S., Luo, X., and Wang, J. (2018) "Rapid and efficient production of cecropin A antibacterial peptide in Escherichia coli by fusion with a self-aggregating protein", BMC Biotechnol. 18, 62.
4.3 Lin, Z., Zhao, Q., Zhou, B., Xing, L., and Xu, W. (2015). Cleavable Self-Aggregating Tags (cSAT) for Protein Expression and Purification. In Insoluble Proteins: Methods and Protocols, E. García-Fruitós, ed. (New York, NY: Springer New York), pp. 65– 78.
4.4 Part:BBa K1230001 - parts.igem.org. http://parts.igem.org/Part:BBa_K1230001
4.5 Part:BBa K1104301 - parts.igem.org. http://parts.igem.org/Part:BBa_K1104301
4.6 Cecropin A. https://www.medchemexpress.com/Cecropin_A.html?locale=ja-JP
4.7 Tobias, J.W., Shrader, T.E., Rocap, G., and Varshavsky, A. (1991) "The N-end rule in bacteria", Science 254, 1374– 1377.
5.1 Florack, D.E.A., Stiekema, W.J., and Bosch, D. (1996) "Toxicity of peptides to bacteria present in the vase water of cut roses", Postharvest Biol. Technol. 8, 285–291.
5.2 Mancinelli, R.L., and Shulls, W.A. (1978) "Airborne bacteria in an urban environment", Appl. Environ. Microbiol. 35, 1095–1101.
5.3 Simek, K., Pernthaler, J., Weinbauer, M.G., Hornák, K., Dolan, J.R., Nedoma, J., Masín, M., and Amann, R. (2001) "Changes in bacterial community composition and dynamics and viral mortality rates associated with enhanced flagellate grazing in a mesoeutrophic reservoir", Appl. Environ. Microbiol. 67, 2723–2733.
5.4 Tenover, F.C. (2009). Antibiotic Susceptibility Testing. In Encyclopedia of Microbiology (Third Edition), M. Schaechter, ed. (Oxford: Academic Press), pp. 67–77.
6.1 Hoppen, C., Müller, L., Albrecht, A.C., and Groth, G. (2019) "The NOP-1 peptide derived from the central regulator of ethylene signaling EIN2 delays floral senescence in cut flowers", Sci. Rep. 9, 1287.
6.2 Nakagawa, Y., Nishikawa, B., and Miyagawa, H. (2021) "Effects of brassinolide on the growing of rice plants", J. Pestic. Sci. 46, 274–277.
4.2 Wang, M., Zheng, K., Lin, J., Huang, M., Ma, Y., Li, S., Luo, X., and Wang, J. (2018) "Rapid and efficient production of cecropin A antibacterial peptide in Escherichia coli by fusion with a self-aggregating protein", BMC Biotechnol. 18, 62.
4.3 Lin, Z., Zhao, Q., Zhou, B., Xing, L., and Xu, W. (2015). Cleavable Self-Aggregating Tags (cSAT) for Protein Expression and Purification. In Insoluble Proteins: Methods and Protocols, E. García-Fruitós, ed. (New York, NY: Springer New York), pp. 65– 78.
4.4 Part:BBa K1230001 - parts.igem.org. http://parts.igem.org/Part:BBa_K1230001
4.5 Part:BBa K1104301 - parts.igem.org. http://parts.igem.org/Part:BBa_K1104301
4.6 Cecropin A. https://www.medchemexpress.com/Cecropin_A.html?locale=ja-JP
4.7 Tobias, J.W., Shrader, T.E., Rocap, G., and Varshavsky, A. (1991) "The N-end rule in bacteria", Science 254, 1374– 1377.
5.1 Florack, D.E.A., Stiekema, W.J., and Bosch, D. (1996) "Toxicity of peptides to bacteria present in the vase water of cut roses", Postharvest Biol. Technol. 8, 285–291.
5.2 Mancinelli, R.L., and Shulls, W.A. (1978) "Airborne bacteria in an urban environment", Appl. Environ. Microbiol. 35, 1095–1101.
5.3 Simek, K., Pernthaler, J., Weinbauer, M.G., Hornák, K., Dolan, J.R., Nedoma, J., Masín, M., and Amann, R. (2001) "Changes in bacterial community composition and dynamics and viral mortality rates associated with enhanced flagellate grazing in a mesoeutrophic reservoir", Appl. Environ. Microbiol. 67, 2723–2733.
5.4 Tenover, F.C. (2009). Antibiotic Susceptibility Testing. In Encyclopedia of Microbiology (Third Edition), M. Schaechter, ed. (Oxford: Academic Press), pp. 67–77.
6.1 Hoppen, C., Müller, L., Albrecht, A.C., and Groth, G. (2019) "The NOP-1 peptide derived from the central regulator of ethylene signaling EIN2 delays floral senescence in cut flowers", Sci. Rep. 9, 1287.
6.2 Nakagawa, Y., Nishikawa, B., and Miyagawa, H. (2021) "Effects of brassinolide on the growing of rice plants", J. Pestic. Sci. 46, 274–277.
ssrA tag mutagenesis
Please also refer to Engineering Success for the design: Engineering Success
Key achievements
We mutagenized ssrA tag by PCR with mixed primers, and successfully obtained a mutant collection
of
engineered ssrA tag derivatives which show various protein degradation efficiencies.
Plasmid construction
First, we tried to obtain the backbone and insert fragments needed for the plasmid
construction.
Backbone fragment was PCR-amplified from pSB4K5-J04450.
The size of the desired band is 3584bp. Each PCR reaction was checked on agarose gel, and the one amplified from 1.0ng of the template was chosen and column-purified because of low background.
Insert fragment was obtained by 2 steps of PCR. The first PCR was performed on the GFP insert derived from BBa_K584001 to add the first 24-bp sequence of ssrA tag, which is not the target for mutagenesis.
The size of the desired band is 798bp. Each PCR reaction was checked on agarose gel, and the one amplified from 0.01ng of the template was chosen.
The first PCR product was then diluted 1:1000 and used as the template for the second PCR to fuse and mutagenize the 9-bp target sequence for mutagenesis with mixed primers.The primers LAA, AAV, ASV, and XXX were used individually, but XAA, LXA, and LAX were pooled and used in the same reaction.
The size of the desired band is 798bp. Each PCR reaction was checked on agarose gel and column-purified.
The column-purified backbone and insert fragments were assembled by InFront Assembly (a homology arm-mediated seamless cloning method like In-Fusion), and transformed into E.coli competent cells.
Backbone fragment was PCR-amplified from pSB4K5-J04450.
The size of the desired band is 3584bp. Each PCR reaction was checked on agarose gel, and the one amplified from 1.0ng of the template was chosen and column-purified because of low background.
Insert fragment was obtained by 2 steps of PCR. The first PCR was performed on the GFP insert derived from BBa_K584001 to add the first 24-bp sequence of ssrA tag, which is not the target for mutagenesis.
The size of the desired band is 798bp. Each PCR reaction was checked on agarose gel, and the one amplified from 0.01ng of the template was chosen.
The first PCR product was then diluted 1:1000 and used as the template for the second PCR to fuse and mutagenize the 9-bp target sequence for mutagenesis with mixed primers.The primers LAA, AAV, ASV, and XXX were used individually, but XAA, LXA, and LAX were pooled and used in the same reaction.
The size of the desired band is 798bp. Each PCR reaction was checked on agarose gel and column-purified.
The column-purified backbone and insert fragments were assembled by InFront Assembly (a homology arm-mediated seamless cloning method like In-Fusion), and transformed into E.coli competent cells.
Imaging bacterial overnight culture under blue light
As a result of transformation, colonies of various fluorescence intensity were obtained on the
LB
plates.. 72 colonies in total were picked from plates, and cultured overnight in LB media. The
image
of E.coli cultures were taken under the blue light.
Bacterial cultures of various GFP intensity were observed.
A10: LB media only
B10: E.coli culture not expressing GFP
B7: E.coli culture expressing non ssrA-tagged GFP
C7: E.coli culture expressing WT ssrA-tagged GFP
B10: E.coli culture not expressing GFP
B7: E.coli culture expressing non ssrA-tagged GFP
C7: E.coli culture expressing WT ssrA-tagged GFP
Bacterial cultures of various GFP intensity were observed.
Sequences and GFP intensity of mutant ssrA tags
Plasmid was extracted from each bacterial culture by miniprep, and then Sanger-sequenced to
identify ssrA tag sequence. Furthermore, to quantify protein degradation efficiency of each
mutant, GFP Fluorescence of each bacterial overnight culture was imaged on Typhoon 9410 (GE
healthcare).
The mutant ssrA tag sequence of each clone was sequenced, and biological triplicate of each
bacterial overnight culture was imaged for GFP fluorescence, including controls ( LB media only,
bacterial culture not expressing GFP (no GFP), and bacterial culture expressing non ssrA-tagged
GFP (no ssrA tag)).
The fluorescence of each culture was quantified by ImageJ as inverted mean gray value. Measured
value of each culture was then normalized to the level of the culture expressing non ssrA-tagged
GFP (no ssrA tag), and presented as mean ± SD from three biological replicates. WT (LAA) and two
mutants reported in a previous research are shown in red boxes.
Plasmid construction
Design
To produce biomaterials more efficiently and environmentally sustainable, we adopted a system of
cell differentiation and division of roles named “asymmetric plasmid partitioning." We
constructed plasmid A required for this system.
Plasmid A has iRFP and superfolder GFP. We used BBa_K515005 for the superfolder GFP controlled
by cI repressor and TetR, this plasmid act as a reporter of asymmetric plasmid partitioning.
Construction
We amplified inserts and backbone fragments by PCR.
All PCR products looked correct, so we purified them with columns. All dsDNA concentration was
enough
(pSB1A3 : 93.2 ng/ul, iRFP : 44.2 ng/ul, oligo_A : 44.2 ng/ul, superfolder GFP : 99.4 ng/ul). To
reduce the
number of inserts, we fused inserts by OE-PCR.
Both of them look correct. We purified OE-PCR1 because its band looks clearer. dsDNA
concentration of
that was 155.6 ng/ul. We got all fragments for plasmid A, so we assembled them (mistakenly we
used iRFP
instead of superfold GFP). After that, we transformed them to DH5a and incubated them overnight.
The
plates after incubation are shown below.
Too many bacteria were cultured, so we were not able to separate each colony. This cause may
inactivate ampicillin, or there was some contamination. To solve these failures, we purified
them by
gel purification. After gel purification, the dsDNA concentration of pSB1A3 and superfolder GFP
were
enough. (pSB1A3 : 12.1 ng/uL, superfolder GFP : 27.6 ng/uL) But that of OE-PCR1 was too low. So
after
this, we purified fragments only by gel purification. We retried, dsDNA concentration of the
dsDNA
concentration of RFP was enough. (15.9 ng/uL) So, we retried assembly and transformation. The
plates
after incubation are shown below.
We picked eight colonies each from plasmid A and A control and amplified them by colony PCR. We
used
two sets of primers, one set covering all of the inserts and the other covering a part of the inserts.
lane 1: pSB1A3-mRFP. The band looked correct (2155bp).
lane 1 and 6: iRFP. The bands looked correct. (1009bp)
lane 1: oligo_A. The bands looked correct. (137bp)
lane 1: superfolder GFP. The band looked correct. (789bp)
lane 1: OE-PCR1 (iRFP and oligo_A, 1116bp), lane 2: OE-PCR2 (oligo_A and superfolder GFP, 896bp)
From the left to the right, plasmid A (×1/10), plasmid A (×1), plasmid
A control (only iRFP and pSB1A3 fragments)
from the left to the right, plasmid A (×1/10), plasmid A (×1),
plasmid A control (only superfold GFP
and pSB1A3 fragments).
lane 1-8: plasmid A (primer: covering all of the inserts), lane 9-16: plasmid A control (primer: covering all of the inserts), lane 17-24: plasmid A (a part of the inserts)
lane 25-32: plasmid A control (a part of the inserts)
1, 3 lanes looked correct (2146 bp). And 17, 19 lanes looked correct (1620 bp), so we read the
sequence of them. After the miniprep, the concentrations of plasmid were 109.3 ng/µl and 139.3
ng/ul. As a result of the reading sequence, both of the plasmids have the correct sequence.So,
we succeeded to construct plasmid A.
References
7.1 Djurhuus, A., Port, J., Closek, C.J., Yamahara, K.M., Romero-Maraccini, O., Walz, K.R.,
Goldsmith, D.B., Michisaki, R.,
Breitbart, M., Boehm, A.B., et al. (2017) "Evaluation of Filtration and DNA Extraction Methods
for Environmental DNA
Biodiversity Assessments across Multiple Trophic Levels", Frontiers in Marine Science 4,
314.
7.2 Flynn, J.M., Levchenko, I., Seidel, M., Wickner, S.H., Sauer, R.T., and Baker, T.A. (2001) "Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis", Proc. Natl. Acad. Sci. U. S. A. 98, 10584–10589.
7.2 Flynn, J.M., Levchenko, I., Seidel, M., Wickner, S.H., Sauer, R.T., and Baker, T.A. (2001) "Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis", Proc. Natl. Acad. Sci. U. S. A. 98, 10584–10589.